The global burden of hepatitis C virus (HCV) infection is at 185 million with an annual rate of 3?4 million new infections each year. In the US, HCV infection is increasing in young adults because of injection drug use. A preventive vaccine is needed in spite of major advances in the development of direct acting antivirals (DAAs) for the treatment of HCV infections. Cumulative evidence has established that both T and B cell immunity contribute to the control of acute HCV infection. The focus of this application is on a B cell-based vaccine that has been technically challenging because of high variability in the viral envelope E1E2 glycoproteins, the natural target of protective antibodies. We have shown that the immunogenic regions on the E2 envelope glycoprotein of HCV segregate into five epitope clusters, designated antigenic domains A?E, and hypervariable region 1 (HVR1). Antigenic domain A elicits non-virus-neutralizing (non-Vn) antibodies and HVR1 elicits isolate-specific virus-neutralizing (Vn) antibodies associated with viral escape. By contrast, antigenic domains B, D, and E elicit Vn antibodies that are more broadly Vn among the major HCV genotypes, and some of their epitopes are not associated with viral escape. Based on these findings, and on recent evidence that the conformational stability of epitopes is a key determinant of immunogenicity, the following questions will be addressed: Does increasing the conformational stability of Vn epitopes within antigenic domains D and E increase the immunogenicity of these conserved epitopes? Can non-Vn antigenic domain A epitopes be silenced in order to decrease the immunogenicity of these decoy epitopes? We will use new X-ray crystallographic information on the E2 glycoprotein, and on human monoclonal antibodies bound to E2 Vn epitopes, to engineer E2 variants for immunological characterization to address these questions. Our objectives are two-fold. First, we will stabilize antigenic domain E (Aim 1) and D (Aim 2) epitopes that mediate broad virus neutralization against diverse HCV genotype and subtype isolates, and that are not associated with viral escape. Second, we will down-modulate antigenic domain A epitopes associated with non-neutralizing antibodies that serve as immunogenic decoys (Aim 3). Structure-guided computational modeling will be employed to design these modifications. E2 variants bearing these mutations will be characterized biochemically and immunologically, followed by evaluation of their capacity to induce Vn antibodies in mice. If successful, these studies will contribute to the development of a rationally designed HCV vaccine to induce broadly Vn antibodies to prevent HCV infection of different genotypes and subtypes.